The plasma-membrane Ca2+ pump or ATPase (PMCA) participates in Ca2+ extrusion from hair bundles. Genetic experiments have established that one PMCA isozyme, PMCA2, plays an essential role in auditory and vestibular function. An underlying assumption of the proposal is that PMCA2 is critical for hearing and balance precisely because of its Ca2+ pumping role in stereocilia. The applicant proposes to identify hair-bundle PMCA, determine its impact on bundle ion balance and energy metabolism, and provide more direct evidence of PMCA's role in hair-bundle function. Four isozymes and dozens of splice forms of PMCA exist, which vary in their biochemical properties. One goal is to identify the isozyme and splice variant of PMCA present in frog, rat, and mouse hair bundles. To identify frog hair-bundle PMCA, the applicant will screen a frog-sacculus cDNA library and isolate PMCA clones. Specific antibodies will be generated to determine which forms reside in bundles using immunocytochemical and immunoprecipitation experiments. Its role in bundle function will be determined quantitatively by expressing hair-bundle PMCA and measuring biochemically and electrophysiologically its pumping properties, including transport rate and affinities for Ca2+, ATP, and calmodulin. PMCA transports in one H+ and hydrolyzes one ATP for every Ca2+ pumped out; therefore, another series of experiments will examine how hair bundles respond to H+ influx and ATP hydrolysis. pH-sensitive dyes will be used to determine the transporters that are targeted to bundles, and it will be determined whether hair bundles possess unique isozymes of creatine kinase or adenylate kinase, two enzymes that serve to maintain high ATP levels. Finally, the role of hair-cell PMCA in auditory function will be tested by re-expressing the hair-bundle splice form of PMCA2 selectively in hair cells of mice lacking a functional PMCA2 gene. These mice, which should only express PMCA in their hair cells and may target PMCA2 exclusively to their hair bundles, will be examined for auditory and vestibular function, using techniques such as auditory-brainstem recording,, distortion-product otoacoustic emissions, electrically evoked otoacoustic emissions, and cochlear microphonics. If re-expression of PMCA2 in hair cells rescues auditory function in these otherwise PMCA2-deficient mice, the key location of PMCA2 in the ear must be the hair cell.